Influenza, an old world virus is responsible for unpredictable periodic pandemics and seasonal outbreaks, and imposes severe burden on public health. With time, geographic spread of influenza virus has increased. Most importantly, cross-species infective capacity of this virus has led to emergence of newer strains from random gene assortments (1). This peculiar feature categorizes it as one of the deadliest virus. In absence of a generic broad spectrum therapy against influenza strains, different approaches are being formulated including using viral components as vaccine, molecules capable of derailing essential pathways of this virus, besides screening of small molecules based on sialic acid moieties to act as receptor mimics. Interestingly, though influenza is known to evolve via gene reassortment to evade immune system and drugs reactive to its surface components, but host cell entry protocol of this old world virus has remained unchanged (2). A pivotal point in the different events in the influenza infection, endosomal entry, fusion with host membranes, integration with host genetic material for replication and packaging of pro-viral particles for further infection is the low pH induced opening of the HA trimer. HA, an envelope protein on viral surface, is packed as a trimer which opens-up upon sensing low pH, an essential event for viral entry (3). The functional relevance of HA entails it to be a better therapeutic target than the other surface expressed viral molecules. But being variable in nature, the molecules and/or antibodies developed against HA exhibit strain dependence (4).
Importantly, structural data available for HA has remained devoid of the information of carbohydrate/sugar moieties associated with this molecule (2). Electron microscopic details with intact virus suggest 25-35% occupancy of the total viral surface by HA. HA glycoprotein exists as spikes of approximate length 137 Å (5). Functionally, HA belongs to class I fusion protein and type I TM class. Class I fusion protein defines as fusion mediated by conformational changes in the protein and type I TM class represent receptor binding as well as fusion ability of the protein (6). Initially, HA is expressed as an inactive precursor molecule, HAO, which is then converted into active, fusion prone pH sensitive molecule by the host enzyme (7). Intriguingly, nature has encoded dual activity in HA, pH sensing based activation and binding to receptor, and to overcome two barriers before releasing its genetic material into host cell (8). On encountering low pH, the inner part of the HA molecule undergoes loop to helix transition resulting in dislocation of the fusion peptide by ˜100 Å towards target membrane for effective viral fusion (3). Irrespective of the fact that HA is a glycosylated entity and is crucial for viral entry, none of the structural studies provides information of the physiologically relevant glycosylated version. The lack of structural information related to glycosylated HA limits the development of anti-influenza drugs/molecules which can target influenza virus via HA molecule.
In current scenario, the evolution of drug resistant strains against FDA approved drugs is alarming (9,10). To overcome the barrier of strain dependence and fast pace of viral evolution there is a need to develop newer target sites and drug molecules. Status of anti-flu drug development with respect to HA has pitfalls like use of monomer of the molecule as template, use of non-glycosylated crystal structure as start point for drug design/screening and strain dependence (4). To overcome these barriers a novel target site needs to be identified in glycosylated HA molecule. Details in the field of anti-influenza therapy clearly suggests the need for identification of a target site in the glycosylated HA molecule and for small molecules capable of blocking the shape changes responsible for viral-host membrane fusion. The invention disclosed here provides a way to fulfill these needs.